ES2678695T3 - Methods and compounds to increase the sensitivity of botulin assays - Google Patents

Abstract

A method of detecting the presence of a botulinum toxin, comprising: providing an assay with a cell that has (a) a molecule with a cleavage site that interacts with a portion of the botulinum toxin and (b) a reporter that provides a signal observable after excision of the molecule at the site of excision induced by botulinum toxin, characterized in that the reporter comprises a fluorescent protein; and incubating the cell in a culture medium containing an isoquinolinyl compound effective to increase the sensitivity of the assay by at least 0.5 log relative to a sensitivity of the baseline provided by the assay using incubation in the absence of said compound; treating the cell with an amount of botulinum toxin, then detecting the signal, and correlating the signal with the presence or absence of botulinum toxin, wherein said compound is effective both to inhibit PKC and to induce the formation of neurites.

Description

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DESCRIPTION

Methods and compounds for increasing the sensitivity of botulin assays Field of the invention

The field of the invention is the detection of botulinum neurotoxins (BoNT).

Background of the invention

Botulinum neurotoxins are the most lethal substances known, and depending on the serotype, the estimated lethal human dose is in the range of 1 to 3 ng of toxin per kg of body weight. Due to their ease of purification and high potency, BoNTs represent a real and potential threat to their use as biological weapons. In addition, BoNTs are increasingly used for cosmetic and therapeutic purposes. Currently, most of the BoNT detection and potency measurements completed in government and industrial laboratories are carried out with the use of animal-based tests that have high costs and low accuracy.

The main advantage of using cell-based assays (CBA) for measurements of BoNT potency is that CBA systems closely mimic the individual physiological stages that occur in neurons during intoxication. BoNTs are zinc dependent endopeptidases composed of a heavy chain, responsible for specific neuronal receptor binding and cell entry, and a catalytic light chain responsible for cleavage of the synaptic protein. Upon entering the neurons, the BoNT specifically break down the protein machinery responsible for the fusion of synaptic vesicles with the plasma membrane, thus inhibiting the release of neurotransmitters at the postsynaptic junction. Because all physiological stages must be taken into account in CBAs, most tests do not meet the sensitivity requirements for measuring BoNT activity.

Recently, the use of stable model cell lines established for the detection of BoNTs has been described, however, the above CBAs do not meet the sensitivity requirements for the quantification of BoNT pharmaceutical preparations or the detection of BoNT in clinical samples. Presumably, the currently established model cell lines lack the critical neuronal characteristics required for efficient BoNT uptake. A recent solution to increase the sensitivity of BoNT is the use of mouse embryonic stem cells (mESC). Because these cells can differentiate terminally into neurons, they are more sensitive to treatment with BoNT. However, an important drawback of this method is the multiple weeks required to completely differentiate the mESC.

International Patent Publication No. WO 2009/035476 A1 describes a molecular construct comprising a donor tag, an acceptor tag, a linker peptide with a cleavage site sequence and a spacer. In specific embodiments, the linker peptide is a substrate of botulinum toxin that can be recognized and cleaved by botulinum toxin.

International Patent Publication No. WO 2009/114748 A1 describes a serotype A activity test of botulinum toxin based on immunity. To determine the presence or absence of an active BoNT / A in a sample, a-SNAP-25 antibodies are produced that bind to an epitope of the BoNT / A cleavage site from a SNAP-25 cleavage product. With such an antibody, the amount of SNAP-25 cleavage products is measured in a cell line that can capture BoNT / A from a sample.

Therefore, there is an urgent need to develop cell-based assays to evaluate and quantify the potency of samples containing BoNT for the defense of biological weapons, foodborne diseases and therapeutic purposes.

Brief Description of the Invention

According to the present invention, a method according to claim 1 is provided. In preferred embodiments, the compound comprises 1- (5-isoquinolinylsulfonyl) -2-methylpiperazine dichloride (H7). The reporter is preferably an artificial construct expressed in the cell, and the first fluorescent protein is preferably selected from the group consisting of the yellow fluorescent protein (YFP), Citrin, Venus and a YPet protein, and the cleavage site preferably comprises at least one of a protein, motif and mutein of SNARE.

Preferably, the reporter does not engage with the second fluorophore in a manner that produces a significant Forster resonance energy transfer (FRET). In such modalities, a cell can advantageously express an enzyme that facilitates reporter degradation at least ten times faster after excision than before excision, and the presence of botulinum toxin correlates with the reduction of the signal from of a baseline signal. As used in the present description, the term

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"non-FRET tests and constructs" refers to tests and constructs that either (a) have no detectable FRET signal, or (b) that produce a FRET signal with a FRET transfer efficiency of less than 5%, of according to method E1 (the relative fluorescence of the donor in the presence (FDA) and the absence (FD) of the acceptor E1 = 1 - FDA / FD) as described in "FRET or No FRET: A Quantitative Comparison", Claude Berney and Gaudenz Danuser, Biophys J. June 2003; 84 (6): 3992-4010. It is contemplated that any FRET signal having a FRET transfer efficiency of less than 5% is at such an insignificant level that it is impractical to use the signal to provide a quantitative measure of the concentration of a component coupled to FRET.

The split portion that contains the reporter is destroyed or otherwise degraded by the local environment, and the presence of the substance in the investigation is later evidenced by the reduction in the reporter's signal. In the context of this application, it is contemplated that degradation of the protected portion will typically be intracellular in at least one of the two routes, by the ubiquitin-dependent process that directs the proteins to the proteasome, or by the autophagy-lysosomal route. On a route, the proteasome is the enzyme. In lysosomal pathways, it is contemplated that the enzymes of interest are hydrolases, which especially include a family of proteases called cathepsins.

In other aspects of preferred embodiments, the portion containing the cleavable reporter comprises, for example, the yellow fluorescent protein (YFP) as a fluorescent protein. YFP is a genetic mutant of the green fluorescent protein, derived from Aequorea Victoria, and has an excitation peak of 514 nm and an emission peak of 527 nm.

Its use is also contemplated in the portion containing the cleavable reporter that are the closely related proteins Citrin, Venus and YPet. The modifications reduced chloride sensitivity, faster maturation and increased brightness (product of extinction coefficient and quantum yield). Of course, any of the fluorescent proteins mentioned in the present description can be modified to include specific characteristics (eg, spectral) or truncated to a specific size.

After cleavage, the construct is divided into two parts, a portion that contains the reporter that is destroyed or in any other way degrades in the cytosol or other local environment, and a second portion. To normalize the signal detection, that second portion may advantageously include a second fluorescent protein, preferably at an opposite end of the reporter, which can help normalize the assay. The second fluorescent protein can be, for example, Cyan Fluorescent Protein (CFP), mCherry or mStrawberry. The portion containing the reporter does not engage with the second fluorophore so as to produce Forster resonance energy transfer (FRET).

Therefore, before exposure of the construct to BoNT or another cleavage substance, the composition containing the construct (either cell-based or in any other way) exhibits a baseline signal, and then after Exposure shows a reduced signal. For the measurement of YFP degradation, the excited YFP emissions (top, Ex500, Em526) and CFP emissions (center, Ex434, Em470) are collected directly. These emissions are subtracted from the fund afterwards and the YFP emission is divided by the emission of CFP to control cell density and reporter expression in individual cells. That emission ratio (YFP / CFP, lower) is how the test is reported.

Destruction or other degradation of the portion containing the reporter occurs at a much faster rate after exposure to BoNT than preexposure. In preferred embodiments, it is contemplated that destruction or other degradation of the portion containing the reporter occurs at least twice (twice) as quickly after exposure as in pre-exposure, but more preferably the post-exposure rate. It is at least 5 times, at least 10 times, at least 100 times in relation to the pre-exposure speed.

Even in other aspects of the preferred embodiments, the local environment is the cytosol of a living cell. For example, YFP can be used as a C-terminal fluorophore, and CFP can be used as an N-terminal fluorophore. Experimental work has verified that, in the absence of BoNT / A, the YFP can be directly excited and collect the emission. The excitation typically occurs at 505nM and the emission corresponding to 527nM. In the presence of BoNT / A, the reporter is cleaved by releasing a fragment containing YFP. That fragment is degraded by the cell, destroying the YFP and its emission. Thus, BoNT / A activity is detected by measurements in relation to the loss in YFP emission. Therefore, FRET is not required, which avoids all the limits and problems indicated above.

In addition, local environments for the non-FRET construct other than living cells are contemplated, including, for example, cytosol of lysed cells and synthetic media containing one or more enzymes capable of degrading the cleavable fragment when cleaved from the reporter molecule , but unable or much less capable of degrading the cleavable fragment before excising the reporter molecule.

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Various objects, features, aspects and advantages of the subject matter of the invention will become more apparent from the following detailed description of the preferred embodiments, together with the attached drawing figures in which equal numbers represent similar components.

Brief description of the drawings

Figure 1 is a graph showing that the treatment of H7, but not the treatment with calfostine, IBMX, db-cAMP or 8-Br-cAMP, causes an increase in the sensitivity of the BoCell ™ Assay.

Figure 2 are graphs that show that the H7 effect is dose dependent and does not affect cell viability.

Figure 3 is a graph showing the effect of H7 pretreatment (before the addition of BoNT / A). Here, H7 also includes the incubation of BoNT / A.

Figure 4 are the graphs showing that 1) only holotoxin (complete BoNT / A) but not the light chain (BoNT / A fragment) induces a response in the BoCell ™ assay and 2) BoNT / A activity can be blocked by the addition of HcR / A that competes for cell receptors. Thus, BoNT / A responses in the assay occur through natural mechanisms.

Figure 5 is a graph showing that multiple batches of H7 from different manufacturers cause an increase in sensitivity. (There is a report in the literature that some lots of H7 from certain manufacturers are not really H7, thus, the effect with multiple lots was confirmed.)

Figure 6 is a graph showing the response and sensitivity of the assay in the presence of H7 after a 24 and 48 hour incubation with BoNT / A. The detection limits of <3 pM are expected to be possible with trial optimization.

Figure 7A is a schematic of a BoCell ™ construct.

Figure 7B is a schematic of an illustrative assay in which cell-based BoNT / A reporters are used to detect BoNT / A activity through loss of YFP fluorescence.

Figure 8A is an image of BoNT / A induced changes in fluorescence responses.

Figure 8B is a graph showing the fluorescence ratios and BoNT / A sensitivities of cell-based reporters.

Figure 8C is a membrane transfer that shows BoNT / A activity in cells independently of the reporter.

Figure 9 comprises multiple graphs showing the emission data for both YFP degradation and for FRET loss in accordance with the practice of the state of the art from the same cell plates.

Detailed description Sequence sequence (s)

In preferred embodiments, the substance under investigation is a botulinum toxin (BoNT), and the cleavage sequence corresponds appropriately with the substance under investigation. For example, BoNT / A, E and C cleave SNAP-25, and BoNT / B, D, F, G cleave synaptobrevin (Syb), in unique but different sites. BoNT / C also cleaves syntaxin in addition to SNAP-25.

The sequences of contemplated cleavage sites may advantageously comprise (a) a SNARE protein, motif, or mutein. "Muteins" of a protein should be interpreted herein as having at least 30% identity with a corresponding native protein, which includes, for example, compositions having at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% identity with the native protein. Variations of identity may comprise one or more additions, deletions and substitutions. The contemplated muteins include fragments, truncated and fusion proteins.

Transfected cells

The hybrid protein (s) that are formed in the transfected cells preferably include a transmembrane domain, which tends to locate the intracellular vesicles for BoNT / B, D, F and G, and therefore have a substrate. attached to the vesicles. The heavy chain-mediated endocytosis of the BoNT in the transfected cell is followed by the presentation of the light chain on the outer surface of the gallbladder, allowing the activity of

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The light chain protease cleaves the cleavage sequence of the hybrid protein (s), therefore, the portion containing the reporter is cleaved, which is then destroyed or degraded to reduce the signal that is Its testing. The full-length Syb, for example, contains 116 amino acids and is located in the vesicles through a single transmembrane domain.

The hybrid protein (s) that are formed in the transfected cells preferably include a transmembrane domain, which tends to be located in the plasma membrane for BoNT / A, C and E. In some contemplated assays, the domain of membrane anchoring comprises a fragment that contains a palmitoylation site. Suitable membrane anchoring domains are described, for example, in patent document no. US 20060134722 by Chapman.

Although it is especially preferred that the transmembrane domain be the transmembrane domain of the synaptobrevin, mutations (e.g., transitions, transitions, insertions, deletions, inversions, etc.) thereof, and even non-synaptobrevin transmembrane domains are further considered suitable for its use in the present description. Similarly, it should be appreciated that the transmembrane domain can also be replaced by another polypeptide moiety that allows at least a temporary anchoring of the hybrid protein to a membrane so that the rest of the hybrid protein is exposed to the cytosol.

With respect to transfected cells expressing the hybrid protein, it is generally preferred that the cell be stably transfected. However, transient transfection is also contemplated. Even more typically it is preferred that the transfected cell be a neuronal cell. However, numerous other non-neuronal cells (including mammalian, rodent and insect cells, and even yeasts and bacterial cells) are further contemplated in the present description. More typically, the cells will constitutively express that the hybrid protein (s) are, therefore, under the appropriate regulatory elements. In alternative aspects, the expression can also be induced.

According to a preferred embodiment, a recombinant nucleic acid molecule, preferably an expression vector, encoding a BoNT substrate polypeptide and a suitable reporter is introduced into a suitable host cell. One skilled in the art can choose a suitable expression vector, preferably a mammalian expression vector, and will recognize that there is a huge number of choices. For example, the pcDNA series of vectors, such as pCI and pSi (from Promega, Madison, Wisconsin), CDM8, pCeo4. Many of these vectors use viral promoters. Preferably, inducible promoters are used, such as the tet-off and tet-on vectors of Clontech (Mountain View, California).

Many cell line options are suitable as a host cell. Preferably, the cell is of a type in which the respective BoNT exhibits its toxic activities. In other words, the cells preferably show suitable cell surface receptors, or in any other way allow the toxin to translocate into the cell efficiently enough, and allow the toxin to cleave the appropriate substrate polypeptide. Specific examples include primary cultured neurons (cortical neuron, hippocampal neuron, spinal cord motor neuron, etc.); PC12 cells or derived from PC12 cell lines; primary culture chromaffin cells; several cultured neuroblastoma cell lines, such as the murine Neuro 2a cholinergic cell line, human SK-N-SH adrenergic cell line and NS-26 cell line. See for example, Foster and Stringer (1999), Genetic Regulatory Elements Introduced Into Neural Stem and Progenitor Cell Populations, Brain Pathology 9: 547-567.

The coding region for the reporter / cleavage site construct is under the control of a suitable promoter. Depending on the types of host cells used, many suitable promoters are known and readily available in the art. Such promoters can be inducible or constitutive. A constitutive promoter can be selected to direct the expression of the desired polypeptide. Said expression construct can provide additional advantages since it avoids the need to cultivate the expression hosts in a medium containing an inducing substrate. Examples of suitable promoters may be LTR, SV40 and CMV in mammalian systems; lac or trp of E. coli in bacterial systems; polyhedron baculovirus (polh) promoter in insect systems and other promoters that are known to control expression in eukaryotic and prokaryotic cells or their viruses. Examples of strong constitutive and / or inducible promoters that are preferred for use in fungal expression hosts are those promoters that can be obtained from the fungal genes for xylanase (xlnA), phytase, ATP synthetase, subunit 9 (oliC) , triosa phosphate isomerase (tpi), alcohol dehydrogenase (AdhA), .alpha.-amylase (amy), amyloglucosidase (AG - of the glaA gene), acetamidase (amdS) and glyceraldehyde-3-phosphate dehydrogenase (gpd). Examples of strong yeast promoters are those obtainable from the genes of alcohol dehydrogenase, lactase, 3-phosphoglycerate kinase and triosaphosphate isomerase. Examples of strong bacterial promoters include SPO2 promoters as well as promoters of extracellular protease genes.

The hybrid promoters can also be used to improve the inducible regulation of the expression construct. The promoter may additionally include features to ensure or increase expression in a suitable host. For example, features may be conserved regions such as a Pribnow box or a TATA box. The promoter may even contain other sequences to affect (such as to maintain, improve or decrease) the levels of nucleotide sequence expression. For example, other suitable sequences

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include the Shl intron or an ADH intron. Other sequences include inducible elements, such as temperature, chemical elements, stress-inducible elements or light. In addition, suitable elements to improve transcription or translation may be present. An example of this last element is the 5 'TMV signal sequence (see Sleat, 1987, Gene 217: 217-225; and Dawson, 1993, Plant Mol. Biol. 23: 97).

The expression vector may also contain sequences that act on the promoter to amplify the expression. For example, the elements that act in cis SV40, CMV and polyoma (enhancer) and a selectable marker can provide a phenotypic trait for selection (eg, resistance to dihydrofolate reductase or neomycin for mammalian cells or amplicillin / tetracycline resistance for E. coli). The selection of the appropriate vector containing the appropriate promoter and the selection marker are within the level of those skilled in the art.

Preferably, the coding region for the construct is under the control of an inducible promoter. Compared to a constitutive promoter, an inducible promoter is preferable because it allows adequate control of the concentration of the reporter in the cell, therefore, the measurement of changes in the signals is greatly facilitated.

For example, the expression can be controlled with the use of the Clontech Tet-on & Tet-off system (Mountain View, California). Under the control of this promoter, gene expression can be regulated precisely, reversibly and quantitatively. In summary, for the Tet-on system, downstream gene transcription only occurs when doxycycline is present in the culture medium. After transcription for a certain period of time, the culture medium can be changed to reduce doxycycline, thus stopping the synthesis of new reporter proteins. Therefore, there is no history of newly synthesized reporter proteins, and one may be able to see a faster change after treatment with the toxin.

Fluorescent analysis

The fluorescent analysis can be carried out with the use of, for example, a photon count epifluorescent microscope system (containing the appropriate dichroic mirror and filters to monitor the fluorescent emission in the particular range), a photomultiplier counting system. photons or a fluorometer. The excitation to initiate the energy emission can be carried out with an argon ion laser, a high intensity mercury arc lamp (Hg), a fiber optic light source, or other filtered high intensity light source. appropriately for excitation in the desired range. It will be apparent to those skilled in the art that the excitation / detection means can be increased by incorporating photomultiplier means to improve detection sensitivity. For example, the two-photon cross-correlation method can be used to achieve detection on a single molecule scale (see for example Kohl et al., Proc. Nat'1. Acad. Sci., 99: 12161, 2002).

Sensitizer

In a particular embodiment, the 1- (5-isoquinolinylsulfonyl) -2-methylpiperazine (H7) dichloride inhibitor significantly increases the sensitivity of the BoCell ™ model cell line to botulinum neurotoxin type A (BoNT / A).

Pretreatment of BoCell ™ modified neuroblastoma cells with H7 caused a rapid increase in both the length of the neurite and the number of neurites per cell. The effect of H7 was dependent on both time and dose with the maximum effects observed with the treatment of H7 ImM. In addition, this phenotypic change could be "rescued" by eliminating H7. Finally, pretreatment of cells with H7 significantly increased the sensitivity of these cells to treatment with BoNT / A as measured by excision of the SNAP-25 reporter.

H7 also inhibits cAMP and cGMP-dependent kinases, although pretreatment of cells with the HA1004 drug, a selective cAMP and cGMP kinase inhibitor failed to induce morphological changes in neuroblastoma cells, suggesting that the effect is specific for PKC inhibition (1990, JBC article). On the other hand, other molecules known as neuronal cell differentiators, which include some PKA and PKC inhibitors, do not increase the sensitivity of the BoCell ™ assay. The increased sensitivity was not observed with other drugs that were previously shown to induce the formation of neurites: Calfostin C, IBMX, dibutyryl-cAMP, and 8-Bromoadenosine-cAMP.

Thus it is contemplated that inhibition of specific PKC isoforms with selective isoquinolinyl analogs and other drugs can infer similar increases in BoNT / A sensitivity in established model cell lines by inducing neurite formation.

It is further contemplated that the culture media may include one or both of H7 and B27, and that the osmolarity of the culture media may be modified to optimize efficacy with respect to specific botulinum toxins, especially those in Table 1. The osmolarity may adjusted according to known principles, which especially include the modification of the salt and / or sugar content. As used in the present description, the term

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"optimization" means taking measures to improve a desired result, which may or may not actually achieve the best possible result.

Table 1 - Excipient formulations of pharmaceutical products based on BoNT / A. Botox® is manufactured by Allergan Inc. (Irvine, CA), Dysport® by Ipsen (France) and Xeomin® by Merz GmbH (Germany).

 Therapeutic

 Active ingredient per vial Form provided Excipients per vial Concentrations of content in 1 ml

 Botox®

 BoNT / A 100 U Lyophilized powder HSA 0.5 mg, NaCl 0.9 mg BoNT / A 100 U, 0.05% HSA, 15.4 mM NaCl

 Dysport® / Reloxin®

 BoNT / A 500 U Lyophilized powder HSA 125 gg, lactose 2.5 mg BoNT / A 500 U, 0.0125% HSA, 0.25% lactose

 Xeomin®

 BoNT / A 100 U Lyophilized powder HSA 1 mg, sucrose 4.7 mg BoNT / A 100 U, HSA 0.1%, sucrose 0.47%

Experimental sensitizer results

The discovery is that treatment of BoCell ™ cells with compound H7 increases the sensitivity of the BoCell ™ assay. From the point of view of the methods, the possible technological advantages of treatment with H7 include:

1. Short test times. With the treatment with H7, sensitivities were obtained with BoNT / A from BoCell ™ after 24 hours of treatment with BoNT / A which are equivalent to the treatments with BoNT / A of 72 hours with the current test conditions. A reduction of the test time of 48 hours.

2. General increase in the sensitivity of the assay. Using H7 we can increase the sensitivity of the assay by 0.5 log - 1.0 log with a 48-hour BoNT / A treatment compared to the current test conditions. It is expected that sensitivity will be further increased with trial optimization.

3. Increases the sensitivity of neuronal cells to other BoNT serotypes. This is currently being tested.

Thus it is contemplated that the use of the compounds contemplated in the present description can increase the sensitivity of the assay by at least 0.5 log, at least 0.6 log, at least 0.7 log, at least 0.8 log, at minus 0.9 log and at least 1.0. Unless the context dictates otherwise, all intervals established in this description should be interpreted as inclusive of their endpoints and open intervals should be interpreted to include only commercially practical values. Similarly, all lists of values should be considered as inclusive of intermediate values unless the context indicates otherwise.

Figures 1-6 show the data generated with the use of H7.

In Figure 1, BoCells were seeded and pretreated for 24 hours with A) H7, B) Calfostin C, C) IBMX, D) dibutyryl-cyclic AMP (db-cAMP), or E) 8-brom o-AMP cyclic (8-Br-cAMP) in the indicated concentration. Control cells were treated with the vehicle equivalent to the maximum concentration of drug tested. After pretreatment, BoCells were treated with BoNT / A containing the indicated drug concentration for an additional 24 hours. The test response was collected by reading the emission at 485 and 535 nm with the use of a Tecan ™ Infinite 500 series microplate reader. The emission ratios (YFP / CFP) were plotted as a function of the BoNT / concentration. TO. The data represent the mean ± standard deviation of the average of the samples processed in triplicate.

In Figure 2, A) the BoCells were seeded and pretreated for 24 hours with the indicated concentration of H7. Control cells were treated with the vehicle equivalent to the maximum concentration of drug tested. After pretreatment BoCells were treated with BoNT / A containing the indicated drug concentration for an additional 24 hours. The test response was collected by reading the emission at 485 and 535 nm with the use of a Tecan ™ Infinite 500 series microplate reader. The emission ratios (YFP / cFp) were plotted as a function of BoNT / concentration. TO. The data represent the mean ± standard deviation of the average of the samples processed in triplicate. B) BoCells were stained with trypan blue after 3x fields of 50 or more cells were counted per treatment and cell viability was calculated.

In Figure 3, BoCells were seeded and pretreated for any of the 3. 6. 12 or 24 hours with 0.75 mM H7. After pretreatment BoCells were treated with BoNT / A containing 0.75 mM H7 for an additional 24 hours. The test response was collected by reading the emission at 485 and 535 nm with the use of a Tecan Infinite 500 series microplate reader. The emission ratios (YFP / CFP) were plotted as a function of the BoNT / A concentration. . The data represent the mean ± standard deviation of the average of the samples processed in triplicate.

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In Figure 4, A) the BoCells were plated and pretreated for 24 hours with 0.75 mM H7. After pretreatment The BoCells were treated with either BoNT / A holotoxin or BoNT / A light chain containing 0.75 mM H7 for an additional 24 hours. B.) The BoCells were pretreated for 24 hours with 0.75 mM H7. 10 mM HcR / A or vehicle was added to the wells for 1 hour before the addition of BoNT / A. The test response was collected after 24 hours reading the emission at 485 and 535 nm with the use of a Tecan Infinite 500 series microplate reader. The emission ratios (YFP / CFP) were plotted as a function of concentration from BoNT / A. The data represent the mean ± standard deviation of the average of the samples processed in triplicate.

In Figure 5, BoCells were pre-incubated with 0.75 mM H7 from three independent manufacturers (listed) for 24 hours after they were treated with BoNT / A in the presence of 0.75 mM H7. The test response was collected after 24 hours reading the emission at 485 and 535 nm with the use of a Tecan ™ Infinite 500 series microplate reader. The emission ratios (YFP / CFP) were plotted as a function of the concentration of BoNT / A. The data represent the mean ± standard deviation of the average of the samples processed in triplicate.

In Figure 6, BoCells were pre-incubated with 0.75 mM H7 for 24 hours after they were treated with BoNT / A in the presence of 0.75 mM H7. The test response was collected after 24 hours and 48 hours by reading the emission at 485 and 535 nm with the use of a Tecan ™ Infinite 500 series microplate reader. The emission ratios (YFP / CFP) were plotted as a function of BoNT / A concentration. The data represent the mean ± standard deviation of the average of the samples processed in triplicate.

Non-FRET based tests

Figure 7A represents the BoCell ™ A BoNT / A BioSentinel construct. The reporter fluorophore, YFP, and the normalization fluorophore, CFP, are coupled by a cleavage sequence, SNAP-25 (green). SNAP-25 palmitoylation locates the reporter to a plasma membrane.

Figure 7B depicts an illustrative test in which reporters based on BoNT / A cells are used to detect BoNT / A activity by loss of YFP fluorescence. Here, the YFP portion is directly excited which leads to fluorescence emission in the absence of BoNT / A. Excision of the reporter by BoNT / A releases a C-terminal reporter fragment that contains the YFP portion in the cytosol. The fragment degrades rapidly and thus the YFP emission is lost. The CFP signal is still used to control the expression levels of the cell-to-cell reporter and cell density.

Surprisingly, not all YFP-related fluorescent proteins are effective as a reporter fluorophore. For example, Figures 8A - 8C provide evidence that reporters containing YFP or the closely related Venus derivative can detect BoNT / A activity in cells, but not mCherry or mStrawberry. Here, Neuro2A cells were grown in a 96-well plate with a 70% confluence and transiently transfected with the use of Lipofectamine 2000 (Invitrogen ™), with reporters containing the indicated N-terminal and C-terminal fluorophores pairs (N-term / C-term). After 24 hours, the cells were incubated in the presence or absence of 10 nM BoNt / A at 37 ° C for 72 hours in 100 µl of phenol red free MEM medium.

Figure 8A shows the changes induced by BoNT / A in fluorescence responses. Semi-automated fluorescence measurements of YFP and CFP were performed with the use of a Nikon ™ TE2000-U fluorescent microscope with 20x magnification and Nikon NIS Elements 3.4 software. Randomly selected pseudo-colored fields are shown for the C-terminal / N-terminal fluorescent protein (FP) fluorescence ratio. The ratios were calculated from the emissions collected after the direct excitation of each fluorophore.

Figure 8B depicts the fluorescence ratios and BoNT / A sensitivities of cell-based reporters. 30 randomly selected cells were analyzed per condition for fluorescence ratios in the presence or absence of 10 nM BoNT / A. The average signal of the 30 cells of 5 microscopic fields in 3 different wells is shown. Cells exhibiting supersaturated fluorescence were excluded.

Figure 8C is a membrane transfer showing that BoNT / A was active in the cells independently of the reporter. All reporters show some cleavage in the presence of BoNT / A, and all native SNAP25 are cleaved. The cells were transfected and treated with BoNT / A as described above, but expanded in 6-well plates. After 72 hours of incubation with BoNT / A, the cells were washed 3X with serum-free MEM, collected by scraping and lysed with the use of M-Per Lysis Buffer (Pierce ™). 40 pg of cell lysate were subjected to SDS-PAGE prior to transfer to nitrocellulose paper and immunoelectrotransfer analysis with the use of an antibody directed against SNAP-25 (clone 71.2, Synaptic Systems). The arrows indicate the position of the full length (closed) and split (open) forms of the reporters. Native SNAP-25 are indicated throughout their length (*) and split (**).

The subject of the invention can extend beyond the cleavable substrates, to any test that has a construct with a reporter that can be unprotected, and then degraded in some way by the cytosol or other

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local environment For example, a susceptible reporter could be modified to include a 'decoy' domain that is used to screen against a library of recombinant proteins that could possibly bind to the decoy domain. Without the decoy domain protected by a binding protein, the susceptible reporter will degrade. In such an assay, the cells expressing the binding proteins will form a complex to protect the reporter susceptible to degradation, while the cells expressing a lure binding partner will illuminate. The decoy domain could advantageously be a small peptide, and the binding partners could be members of a protein library (or protein mutants). The system could also be reversed so that there is a library of tested decoy domains against a single test protein (or test protein library).

In each of these cases it is considered advantageous to include a second reporter that does not degrade after exposure by the cytosol or other local environment, or is degraded at least much more slowly after exposure than the first reporter.

Furthermore, while the reporter can be conveniently selected from suitable fluorophores, it is contemplated that the reporter could be replaced or augmented by any other protein or other component with a defined function that is known to (a) have a relatively rapid turnover in the unprotected cell, and (b) that can be protected by interaction with a binding partner. Defined functions include transcription activators for the reporter gene, repressors of lethal genes, etc. (anything that can be easily identified or selected).

Figure 9 comprises multiple graphs showing the emission data for both YFP degradation and for FRET loss in accordance with the practice of the state of the art from the same cell plates. For YFP degradation, direct and singularly excited YFP emissions (top, Ex500, Em526) and CFP emissions (center, Ex434, Em470) are collected. These emissions are subtracted from the fund afterwards and the YFP emission is divided by the emission of CFP to control cell density and reporter expression in individual wells. That emission ratio (YFP / CFP, lower) is then used for the test report.

For the loss of FRET, FRET emissions (higher, Ex434, Em526) and CFP emissions (center, Ex434, Em470) are collected. These emissions are subtracted from the fund afterwards, and the FRET emission is divided by the emission of CFP to control cell density and reporter expression in individual wells. That emission ratio (FRET / CFP, lower) is shown here to compare with the normal method.

The key comparison is the loss of YFP directly excited based on the loss of FRET emission. From the comparison between the measurements and the corresponding curves, it is immediately apparent that the general dynamic range for YFP degradation is much greater than the dynamic range of FRET emission losses. In some cases, there is no difference, statistically, between cells treated without BoNT as a function of cells treated with saturating concentrations of BoNT when only unprocessed FRET emissions are observed. For the loss of the FRET method, the BoNT dose response only becomes clear after dividing the FRET emission by the CFP (donor) emission. The issuance of CFP (donor) shows a small increase in emission due to deactivation in response to the reporter's excision.

In summary, the loss of the FRET method poorly reports BoNT-induced changes in the reporter, or not at all, and therefore cannot be used for a correct qualitative and quantitative determination. On the contrary, the preferred methods contemplated in the present description have a high degree of specificity and reproducibility, which makes it possible to rely on the data for both qualitative and quantitative analysis.

All terms should be interpreted as broadly as possible in line with the context. In particular, the terms "comprises" and "comprising" should be construed as references to elements, components or stages in a non-exclusive manner, indicating that the elements, components or stages referred to may be present or used, or be combined with other elements, components or stages that are not expressly mentioned. When the claims of the specification refer to at least one of something selected from the group consisting of A, B, C ... and N, the text should be interpreted as requiring only one element of the group, not A plus N, or B plus N, etc.

Claims (9)

1. A method of detecting the presence of a botulinum toxin, which comprises: 5 provide an assay with a cell that has (a) a molecule with a cleavage site that interacts with a portion of the botulinum toxin and (b) a reporter that provides an observable signal upon excision of the molecule at the site of excision induced by botulinum toxin, characterized in that the reporter comprises a fluorescent protein; and incubating the cell in a culture medium containing an isoquinolinyl compound effective to increase the sensitivity of the assay by at least 0.5 log relative to a sensitivity of the baseline provided by the assay using incubation in the absence of said compound; treat the cell with an amount of botulinum toxin, then detect the signal, and correlate the signal with the presence or absence of botulinum toxin, wherein said compound is effective both to inhibit PKC and to induce the formation of neurites. fifteen 2. The method according to claim 1, characterized in that the isoquinolinyl compound comprises 1- (5-isoquinolinylsulfonyl) -2-methylpiperazine dichloride (H7). 3. The method according to claim 1, characterized in that the culture medium comprises B27. twenty 4. The method according to claim 1, characterized in that the osmolarity of the media is optimized for Botox, ie BoNT / A 100 U, 0.05% HSA, 15.4 mM NaCl or it is optimized for Dysport / Reloxin, that is, BoNT / A 500 U, 0.0125% HSA, 0.25% lactose, or it is optimized for Xeomin, that is, BoNT / A 100 U, 0.1 HSA %, 0.47% sucrose. 25 5. The method according to claim 1, characterized in that the reporter is an artificial construct expressed in the cell. 6. The method according to claim 1 characterized in that the reporter comprises the protein The fluorescent and cleavage site, and the cleavage site comprises at least one of a SNARE protein, SNARE protein motif and SNARE protein mutein. 7. The method according to claim 6 characterized in that the molecule includes the reporter and a second fluorophore, 35 preferably where the reporter does not mate with the second fluorophore so that it produces Forster resonance energy transfer (FRET). 8. The method according to claim 1, characterized in that the cell is selected to express a degrading enzyme that facilitates the degradation of the reporter at least twice faster after 40 the split than before the split, or wherein the cell is selected to express a degrading enzyme that facilitates reporter degradation at least five times faster after excision than before excision; or where the presence of botulinum toxin correlates with the reduction of the signal from a baseline signal. Four. Five 9. The method according to claim 1, characterized in that the fluorescent protein is selected from the group consisting of Yellow Fluorescent Protein (YFP), Citrine, Venus and a YPet protein. image 1 [BoNT / A] (M) Control curve (DMBG) B -0 "" Cal C 1 uM image2 [BoNT / A] (M) Figure 1 £ 050 ip) Coniro! - - ♦ -7H 1 mM 79.0 7H 0.1 mM - - || - Control curve (H20) D db-cAMP 1 mM image3 0 1Q-11 10 ~ 10 10-9 10-8 10-7 [BoNT / A] (Vi) image4 image5 or.. Ll IT Ll image6 100 80 ~ o (ü M 60 i 1q (or > 0 40 ■ a 200 Uamr [BoNT / A] (M) Yes "r AND or CM THE OR the CD THE image7 l image8 Figure 4 EC50 (pM) H Holotoxin 130.1 ^ Light chain -— [BoNT / A] (M) image9 EC50 (pM) Ü Holotoxin 256.9 ♦ HcR / A image10 Figure 5

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YFP / CFP image11 [BoNT / A] (M) image12 Figure 7A EMISSION EXCITATION 505nM 527nM image13 Figure 7B EMISSION EXCITATION 505nM 527nM EMISSION EXCITATION 505 nM 527 nM image14 image15 YFP fluorescence disappears image16 * KZ GFP / mStrawberry GFP / mCherry • CFP / YFP Cerulean / V'enus "" T " TT ” CO <N Emission ratio FP C-term / FP N-term QQ oo and .OR) LL <I— z or you i- GFP / mStrawberry GFP / mCherry CFP / YFP Cerulean / Venus * OR oo and .OR) LL image17 ÍBoNT / A] (pM) i £ [BoNT / A] (pM) image18 _a m £ 5 ° í! 00 00